Kinetics of water-insoluble phosphoglycerate mutase - ACS Publications

Coagulation studies with linear copolymers of aliphatic hydrocarbons and maleic acid: New class of anticoagulants. Yeheskel Shamash , Benjamin Alexand...
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BIOCHEMISTRY

Action of Water-Insoluble Trypsin Derivatives on Prothrombin and Related Clotting Factors* Abraham Rimon,t Benjamin Alexander, 1 and Ephraim Katchalski

ABSTRACT: The action of two water-insoluble derivatives of trypsin, IPTT and IMET, on factor I1 (prothrombin) is described. The water-insoluble polytyrosyltrypsin (IPTT) was prepared by coupling polytyrosyltrypsin with a polydiazonium salt derived from a copolymer of p-aminophenylalanine and leucine. The water-insoluble maleic acid-ethylenetrypsin (IMET) was prepared by coupling trypsin with a copolymer of maleic anhydride and ethylene. During interaction with IPTT, factor I1 progressively disappeared while thrombin evolved. Factors VI1 and X present in the factor I1 preparation

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he complex sequence of blood clotting involves several steps which are enzymatic (Macfarlane, 1964; Davie and Ratnoff, 1964). Inert zymogens are converted to active enzymes, which in turn activate other constituents. A notable example is the conversion of factor I1 (prothrombin) to thrombin, a major step in this sequence. There are at least five pathways by which this may be induced: (1) in the course of spontaneous coagulation, via the elaboration of blood (intrinsic) thromboplastin from several sequentially interacting factors; (2) by interaction of factor I1 with tissue thromboplastin, Caz+, and other clotting factors; (3) by certain proteolytic enzymes; (4) by certain anions; ( 5 ) by polylysine and related compounds (for review see Alexander, 1958; Ferguson et al., 1960; Alexander, 1962; Macfarlane, 1964; Davie and Ratnoff, 1964; Miller et al., 1961). To what extent these mechanisms are identical with, or simulate one another, or what fundamental alteration in factor I1 is necessary for its activation, is obscure. Progress in purification of factor I1 and availability of purified proteases led to extensive investigation of the

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* From the Department of Biophysics, The Weizmann Institute of Science, Rehovoth, Israel. Received Augusf 2, 1965; revised November 11, 1965. This investigation was supported by Grant AM-03083 of the National Institutes of Health, U. S. public Health Service; by the Air Force Ofice of Scientific Research. OAR Grant A F EOAR 63-59. through the EuroDean Office, Aerospace Research, U. S. Air Force; and by Grant-HE00656 of the Public Health Service, United States. t From the Department of Microbiology, Tel-Aviv University, Tel-Aviv. 1Commonwealth Fund Fellow on Sabbatical leave from the Yamins Research Laboratory, Beth Israel Hospital, and the Department of Medicine, Harvard Medical School, Boston, Mass. Inquiries regarding this investigation should be made to this author.

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used were concurrently activated. Factor I1 could also be degraded by IMET. In this case, however, no thrombin appeared. Factor X was activated by IMET similarly to IPTT although to a lesser extent. No activation of factor VI1 by IMET occurred. Under the experimental conditions used thrombin could be readily degraded by IMET but not by IPTT or native trypsin. The changes recorded in the biological activity of factor I1 caused by IPTT or IMET were accompanied by corresponding alterations in the electrophoretic patterns of the reaction mixtures.

third pathway, with the view that this approach would help elucidate the structure of this factor and its activation (Alexander and Pechet, 1962). Of many proteolytic enzymes studied, only trypsin and papain produce thrombin from factor I1 (Alexander, 1958). The others were either inert, or they progressively inactivated the zymogen. In contrast to papain, which apparently requires the presence of factor VI1 (proconvertin), factor X (Stuart factor), or both, trypsin produces thrombin directly from factor I1 (Alexander, 1958; Alexander and Pechet, 1958). Because of this, and the well-known ability of trypsin to activate other protease precursors (trypsinogen, chymotrypsinogen, and plasminogen), its effect on factor I1 and other clotting factors was studied intensively (Pechet and Alexander, 1960). The availability of water-insoluble trypsin derivatives provided an additional tool for exploring factor I1 activation. Two derivatives were used : (a) water-insoluble maleic acid-ethylenetrypsin (IMET)' (Levin et af., 1964) in which trypsin is coupled to a copolymer of maleic acid and ethylene; (b) water-insoluble polytyrosyltrypsin (IPTT) (Bar-Eli and Katchalski, 1963) obtained by coupling polytyrosyltrypsin with a polydiazonium salt derived from a copolymer of p-aminophenylalanine and leucine. Since by virtue of their insolubility these trypsin derivatives can be removed at will, they were used to explore various stages of the activation process. It was also deemed of interest to comDare the action of IMET and I m on factors 11, VII, and X, since it has been shown by Levin et al. (1964) that these trypsin derivatives act quite differently on lysozyme. Our results

Abbreviations used: IPTT, water-insoluble polytyrosyltrypsin ; IMET, water-insoluble maleic acid-ethylenetrypsin.

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FIGURE 1 : Changes in concentration of factor 11, VII, X, and thrombin during incubation of a factor I1 preparation with IPTT under the conditions specified in the test.

indicate that in some respects they also act differently on factor I1 and related clotting factors. Materials Factor I1 was prepared from oxalated bovine plasma by the method of Goldstein et al. (1959), and kept lyophilized. The dry powder obtained by lyophilization from a saline solution of the material contained 65% protein with a factor I1 activity of 950 units2 per mg of protein. It also contained some factors VI1 and X activity but was devoid of detectable factor V. Thrombin was obtained from Upjohn Co. Twice-crystallized salt-free lyophilized trypsin was obtained from Worthington Biochemical Corp. IPTT was prepared by coupling polytyrosyltrypsin with a water-insoluble diazonium salt derived from a copolymer of p-amino-DL-phenylalanineand L-leucine (molar residue ratio 1 :l), according to Bar-Eli and Katchalski (1963). The proteolytic activity of the IPTT obtained, containing about 20 mg of protein per 100 mg of water-insoluble enzyme, was assayed on casein as described by Northrop er al. (1948). IPTT (311 mg) had the activity of 1.0 mg of native trypsin. A stock suspen~~

Normal plasma contains approximately 250 unitslml, as determined by the method of Ware and Seegers (1949). 2

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sion of IPTT in 0.1 M phosphate buffer, pH 7.4, possessing a caseinolytic activity of 23 pg of native trypsin per ml of suspension, was prepared and stored at 4". IMET was prepared by coupling trypsin with ethylene-maleic anhydride copolymer, obtained from Monsanto Chemical Co., according to the procedure of Levin et al. (1964). The copolymer to enzyme ratio used was 1 :4 (w/w). The preparations contained about 74 mg protein (Kjeldahl) per 100 mg of the waterinsoluble preparation dried to constant weight at 100". An amount of 197 mg of IMET showed a caseinolytic activity of 1.0 mg of trypsin. A stock suspension of IMET in 0.1 M phosphate buffer, pH 7.4, possessing a caseinolytic activity of 23 pg of soluble trypsin per ml of suspension, was prepared and stored at 4". Methods Assay of Thrombin. This was done by measuring the clotting time with bovine fibrinogen, determined at 37' in glass test tubes (8 X 75 mm) of uniform batch. An amount of 0.2 ml of a 2 % solution of purified fibrinogen (91-98z clottable) (Laki, 1951) in 0.15 M NaCl was mixed with 0.2 ml of the thrombin solution to be tested. The tubes containing the combined ingredients were agitated for 15 sec, and gently tipped from time to time thereafter. The interval, recorded with a stop watch,

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between the admixture and visible solid gelation was taken as the clotting time. The amount of thrombin present was interpolated from a calibration curve obtained under the same conditions with a known amount of thrombin, and was expressed in thrombin units. Assay of Factors ZZ, VZZ, and X . Factor I1 was measured by the two-stage method of Ware and Seegers (1949) as modified by Alexander and Goldstein (1955). Factor VI1 was assayed according to Owren (1953). Factor X was determined according to Bachmann et al. (1958). Activation ofFactor ZZ.A solution of 200 mg of factor I1 in 20 ml of 0.025 M phosphate buffer pH 7.4 was prepared. Four ml was removed for control measurements, and the rest was incubated at 37" with 0.5 ml of the stock suspension of IPTT or IMET. Aliquots were withdrawn at various time intervals, filtered through Whatman No. 1 paper (for IPTT) or through a Millipore filter (Millipore Corp. HA 0.45 b) (for IMET), and the filtrates were assayed for thrombin and factors 11, VII, and X, and subjected to ultracentrifugation and electrophoretic measurements, as described below. Sedimentation Measurements. Sedimentation runs were made in a Spinco Model E ultracentrifuge using a 12-mm Kel-F cell with 4" sector. Temperature was maintained constant during each run in the range of 21-23'.

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RTMON,

BENJAMIN ALEXANDER,

Electrophoresis. Free boundary electrophoresis was performed in a Perkin-Elmer electrophoresis apparatus Model 38 A using the standard 2-ml cell in Verona1 buffer at pH 8.6 and ionic strength 0.1.

Results Enzymatic Studies. Incubation of purified factor I1 with IPTT under conditions given in Methods resulted in thrombin elaboration, as well as marked enhancement in the activities of factors VI1 and X that were present in the preparation (Figure 1). Noteworthy is the early sharp drop in two-stage factor I1 activity some time before the appearance of substantial amounts of thrombin. Under the experimental conditions used, 63% of the prothrombin had disappeared after 10 min of interaction but only trace amounts of thrombin were demonstrable. Thereafter, thrombin progressively appeared. The activities of both factors VI1 and X were greatly enhanced, the latter with an initial value of 33x7,,3attaining a level of ll,OOO% after 220 min of interaction with IPTT. The effect of IMET, incubated with factor I1 under

3 In terms of normal plasma which is arbitrarily defined as containing 100% factors VI1 and X.

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similar conditions, was quite different (Figure 2). Although factor I1 activity declined rapidly as with IFTT, no thrombin could be detected. As with IPTT, IMET also activated factor X, but to a much lesser extent, to a value of 130Oz after 220 min of incubation. In striking contrast to the effect of IPTT, factor VI1 appeared to be unaffected by IMET. Conceivably the early disappearance of factor I1 activity might have been referable to the elaboration of a by-product during conversion of factor I1 to thrombin, which, in turn, might have adversely affected the quantitative assay for thrombin. The following experiment excluded this possibility. Factor I1 (36 mg) was dissolved in 3.0 ml of 0.02 M phosphate buffer, pH 7.0. To this was added IMET sedimented from 2.0 ml of the stock IMET suspension. The mixture was incubated for 1 hr at 37", and then filtered through a Millipore pad (0.22 p). This is referred to as IMET-factor I1 digest. To this was added varying amounts of commercial thrombin (Parke, Davis), and the mixture was assayed for clotting activity of thrombin on fibrinogen (see Methods). As a control, the IMET-factor I1 digest was replaced by saline. No significant difference was observed between the two thrombin-fibrinogen calibration curves correlating thrombin activity with clotting time. The question arose as to whether IMET failed to activate factor 11, or whether it degraded thrombin as fast as it may have evolved. To explore this point, thrombin was incubated for 30 min at 37" with IMET, with IFTT, and with native trypsin, respectively, in amounts equivalent in caseinolytic activity. The results indicated that thrombin was progressively inactivated

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by IMET but not by IPTT or soluble trypsin (Figure 3). If incubation was extended for several hours, or if the enzyme concentration was increased, thrombin inactivation also occurred with both IPTT and native trypsin. Nevertheless, IMET was clearly more effective in this regard. It would thus appear that the failure to detect thrombin after the interaction of factor I1 with IMET could be explained by the prompt destruction of thrombin as it evolved. Indeed, in some experiments where high concentrations of factor I1 were incubated with IMET, trace amounts of thrombin were found. Sedimentation and Electrophoretic Studies. The changes induced in factor I1 by I P m and IMET were followed by observations in the ultracentrifuge and in free boundary electrophoresis. Figures 4 and 5 show the ultracentrifugal patterns of the factor I1 preparations exposed to IPTT or IMET. The preponderant component is reflected in a distinct peak with an average ~ 2 0 value . ~ of 4.57. Adjoining it was a small shoulder representing material that sedimented faster. This pattern did not change appreciably during 4 hr of incubation with either IFTT or IMET, except that in the case of IPTT the sedimentation constant of the major component decreased from 4.57 to 4.24, and in the case of IMET from 4.62 to 4.05. The electrophoretic patterns are shown in Figures 6 and 7. The profile of the starting material, the prothrombin-rich fraction, reveals a main peak exhibiting an electrophoretic mobility of 5.5 X lop5cm2/v/sec, and two lesser components with mobilities of 2.2 x 10-5 cmz/v/sec and 4.1 X cm2/v/sec, respectively. After 12 min of incubation with either enzyme derivative, and more profoundly after 70 min, the electrophoretic

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Sedimentation pattern of untreated factor I1 preparation, and after incubation for different time intervals with IPIT. (A) Control experiment: time of incubation, zero; the figure on the right taken 16 min, that on the left 32 min after rotor reached maximum speed. (B) Incubation with IPTT for 12 min. Figure on the right sedimentation for 16 min, that on the left for 32 min. (C) Incubation with IPTT for 70 min. Right sedimentation for 16 min, left for 32 min. Bar angle 60",rotor speed 59,000 rpm, temperature 20.3". Sedimentation to the left. FIGURE 4:

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pattern had altered completely. With IPIT (Figure 6), 4 peaks were observed with mobilities of 1.9 X (A), 3.9 X 10F @ 5.6 I)X , (C), and 6.1 X lWs cmz/v/sec (D). The mobility of the fastest moving component (D) was identical with that of thrombin prepared from the activation of the same prothrombin preparation with 0.85 M citrate solution (Seegers et a/., 1950), and run under similar conditions. Peak C probably represents the remaining factor lI. Peaks A and B probably reflect contaminating proteins (perhaps factors VI1 and X)which are present in the fraction, and which appear in the starting material. Their areas, however, increased considerably during incubation with the enzyme. With IMET (Figure 9,no elaboration of a fast moving entity is observed. Also as already mentioned, no detectable thrombin appears. Moreover, the area of the original major peak with 5.5 X mobility progressively decreases, the peak of 4.1 X 1W6 disappears, and a new peak appears with a mobility of 3.3 X lW5, which is probably a degradation product derived from factor I1 by IMET.

A R R A H A M RIMON, R E N J A M I N ALEXANDER,

FIGURE 5 : Sedimentation pattern of untreated factor I1 preparations, and after incubation for different time intervals with IMET. Details as for Figure 4.

Discussion On the basis of previous experience with native trypsin, one would expect that the water-insoluble trypsin derivatives would activate factor I1 as well as factors VI1 (Alexander, 1958; Pechet and Alexander, 1960; Alexander et a/., 1962) and X (Pechet and Alexander, 1960; Alexander and Pechet, 1962; Yin,1964; Papahadjopoulos e? a/., 1964). The data are generally in accord witb earlier observations. In scrutinizing the kinetics of factor II activation by IPTT, one is impressed by the fact that biologically demonstrable factor I1 rapidly disappears before a substantial amount of thrombin appears. The same phenomenon has been previously observed with soluble trypsin (Alexander, 1962), and also during the activation of factor I1 preparations by citrate (Seegers et a/., 1950; Alexander, 1962). It further confirms the view that in the transition of factor I1 to thrombin an intermediate substance is produced prior to the appearance of thrombin. This intermediate compound might represent the fast moving component seen in the electrophoretic pattern after 12 min of factor I1 incubation with IF'TT (Figure 6, peak D), possessing the same electrophoretic mobility as thrombin, run under the same conditions. In contradistinction to the results obtained with IPTT, no detectable thrombin evolved on incubating factor I1 with IMET. Since factor I1 disappeared from the reaction mixture (Figure 2), and since thrombin was readily destroyed by IMET (Figure 3), it is reason-

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able to assume that some thrombin was evolved, but it was degraded as fast as it formed. As a matter of fact, when high concentrations of factor I1 were interacted with IMET, traces of thrombin could be detected in the mixture. Nevertheless, thrombin elaboration by IMET at a rate that was sufficiently rapid to assure survival of some of it may have required activation of factor VI1 by IMET, which did not occur. This possibility is supported by the observation that the activation of factor VI1 and X by trypsin enhances thrombin formation from factor I1 (Alexander and Pechet, 1962; Ferguson et al., 1960). Requirement for factor VI1 has already been established for thrombin formation via the citrate activation pathway (Alexander, 1958; Goldstein et af., 1959; Streuli, 1959; Alexander, 1962; Deutsch and Lechner, 1964), or with papain (Alexander, 1958; Alexander and Pechet, 1958). Moreover, prior activation of factor VI1 by trypsin profoundly accelerates subsequent factor I1 conversion by trypsin (Alexander and Pechet, 1961; Alexander, 1962). The electrophoretic patterns of factor I1 treated with IFTT or IMET (Figures 6 and 7 ) clearly reveal the gradual transformation of prothrombin to new molecular entities. No definite assignment can be given at this stage to the various peaks noted. A comparison of Figure 6 with Figure 7 shows, however, that different products are formed on incubation of factor I1 with IPTT or with IMET. It is of interest that the alteration

FIGURE 7 : Electrophoretic patterns of factor I1 preparation before and after incubation with IMET. Details as for Figure 6.

in biological activity and electrophoretic pattern recorded are not accompanied by any marked changes in the sedimentation schlieren profile (see Figures 4 and 5). IPTT and IMET showed striking differences also in their action on factors VI1 and X.Whereas both factors are greatly activated by IPTT, only factor X is activated by IMET, and this to a lesser extent than with IPTT (Figures 1, 2). These noteworthy differences in two preparations derived from the same enzyme are most likely attributable to the different nature of the polymer carriers used for the preparation of these water-insoluble trypsin derivatives. In the case of IPTT the trypsin is bound to a neutral carrier possessing many hydrophobic groups, whereas in IMET the bound trypsin molecules are surrounded at neutral pH by many negatively charged hydrophilic carboxylic groups. It might, therefore, be expected that the affinity of the water-insoluble enzyme derivatives for susceptible amide bonds in a high molecular weight substrate would be different for IMET and IPTT. Peptide bonds of factors 11, VII, and X essential for biologic activity might therefore show different susceptibility to IPTT or IMET. The above consideration might be extended to explain the selective specificity toward high molecular weight substrates of enzymes such as trypsin, thrombin, and plasmin possessing the same activity toward low molecular weight substrates such as tosyl- or benzoylarginyl esters. Assuming identical catalytic sites in these enzymes, one has to postulate that the ultimate speci-

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ficity of the enzymes toward their corresponding protein substrate is predicated upon a high affinity between distinct noncatalytic sites of the enzymes and well-defined regions of the high molecular weight substrates. References Alexander, B. (1958), Fourth International Congress of Biochemistry, Vol. 10, Vienna, Austria, p 37. Alexander, B. (1 962), Thromb. Diath. Haemorrhag. 9, Suppl. 2, 179. Alexander, B., and Goldstein, R. (1955), The Coagulation of Blood. Methods of Study. L. M. Tocantins, Ed., New York, Grune and Stratton, pp 90-97, 141148. Alexander, B., and Pechet, L. (1958), Federation Proc. 17,179. Alexander, B., and Pechet, L. (1962), Proceeding of the VIIIth Congress of European Society of Hematology. VOl. 11, p 404. Bachmann, F., Duckert, F., and Koller, F. (1958), Thromb. Diath. Haemorrhag. 2, 24. Bar-Eli, A., and Katchalski, E. (1963), J. Biol. Chem. 223.1690. Davie; E. W., and Ratnoff, 0. D. (1964), Science 145, 1310. Deutsch, E., and Lechner, K. (1964), Xth Congress

of the International Society of Haematology, pG :19. Ferguson, J. H., Wilson, E. G., Iatridis, S. G., Rierson, H. A., and Johnston, B. R. (1960), J. Clin. &vest. 39, 1942. Goldstein, R., Le Bolloc’h, A. G., Alexander, B., and Zonderman, E. (1959), J. Biol. Chem. 234, 2857. Laki, K. (1951), Aich. Biochem. Biophys. 32; 317. Levin, Y., Pecht, M., Goldstein, L., and Katchalski, E., (1964), Biochemistry 3, 1905. Macfarlane, R. G. (1964), Nature 202, 495. Miller, K. D., Copeland, H. W., and McGarrahaw, J. F. (1961), Proc. SOC.Exptl. Biol. Med. 108, 117. Northrop, J. H., Kunitz, M., and Herriot, R. M. (1 948), Crystalline Enzymes, 2nd ed, New York, Columbia University. Owren, P. (1953), Am. J. Med. 14, 201. Papahadjopoulos, D., Yin, E. T., and Hanahan, D. J. (1964), Biochemistry 3, 1931. Pechet, L., and Alexander, B. (1960), Federation Proc. 19,64. Seegers, W. H., McClaughry, R. I., and Fahey, J. L. (1950), Blood 5, 421. Streuli, F. (1959), Thromb. Diath. Haemorrhag. 3, 194. Ware, A. G., and Seegers, W. H. (1949), J. C h . Pathof. 19,471. Yin, E. T.(1964), Thromb. Diath. Haemorrhag. 12, 307.

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